PROJECT SUMMARY/ABSTRACT: Neuroinflammation propagated by peripherally-derived monocytes/macrophages may have a causative role in chronic anxiety disorders. We report that repeated social defeat (RSD), a preclinical mouse model, induces a sympathetic-mediated release of myeloid cells from the bone marrow that traffic to regions of the brain associated with fear and anxiety. These peripheral myeloid cells promote a robust interleukin (IL)-1 inflammatory signal in the brain that induces prolonged anxiety-like behavior. Recent clinical data support a link between chronic stress, inflammatory monocytes, and anxiety. Critical to this proposal, RSD causes ?stress- sensitization? which is associated with long-term changes in the myeloid cells in the spleen and brain. This is relevant because clinical studies also detect inflammatory monocytes in circulation with stress and observed increased monocyte production in the spleen. Moreover, an important consequence of stress-sensitization with RSD is recurring anxiety after re-exposure to an acute stressor. This grant proposal will focus on stress- sensitization of the myeloid population that establishes in the spleen. The recurrence of anxiety is dependent on the release of inflammatory Ly6Chi monocytes from the spleen, which traffic to the brain and augment neuroinflammation. We report that splenectomy and blockade of norepinephrine (NE) both prevent increased monocyte trafficking to the brain and both block the recurrence of anxiety in stress-sensitized mice. Furthermore, novel data show that RSD causes substantial engraftment of hematopoietic stem progenitor cells (HSPCs) in the spleen. We highlight novel data that HSPCs in the spleen serve as a generator of ?stress- sensitized? myeloid cells that are readily released following acute stress-induced activation of the sympathetic nervous system (SNS). Thus, we hypothesize that RSD promotes the establishment of a unique splenic myeloid population that becomes a critical cellular inflammatory mediator of recurring anxiety-like behavior. To address this hypothesis, three aims are proposed here using RSD in mice. (Pts.3, 4&12) In Aim-1, we will determine the kinetics and transcriptional profiles of splenic monocytes and HSPCs in stress-sensitized mice. (Pts.5, 11&12) In Aim-2, we will determine the sympathetic-dependent stromal cues that facilitate the development of splenic myelopoiesis with stress-sensitization. Several pharmacological and genetic interventions will be used to address the specific contribution of ?-adrenergic receptor activation in the release, establishment, and maintenance of this unique splenic HSPC population. In Aim-3, selective ablation of splenic monocytes will be used to prevent stress-sensitization and block the recurrence of anxiety. Several advanced strategies will be used to do this. Collectively, completion of these aims will provide new insight into long-term sensitization of myeloid cells by RSD and their critical contribution to recurring anxiety.